Tomato CDI as a Strong Chymotrypsin Inhibitor ... - Plant Physiology

following the manufacturer’s instructions. Phagemid-infected E. coli cells were grown
in the presence of 10 mM isopropyl-beta-D-thiogalactopyranoside to induce the
synthesis of the β-galacturonase fusion protein, and upon plaque formation, proteins
were transferred to nitrocellulose membranes. Clones expressing the JIP21 fused to βgalacturonase
were revealed by immunostaining as indicated above. As a control,
membranes containing protein extracts of tomato leaves, which were either treated or
not with 2 mM Methyl-jasmonate and separated by SDS-PAGE, were processed
simultaneously.
The cDNA obtained in the immunoscreening was used as a probe to screen a
tomato genomic DNA library constructed in λ-EMBL (Clontech), and the positive
clones were isolated, purified and characterized, as described in Sambrook et al. (1989).
DNA sequencing was performed on an ABI PRISM DNA sequencer 377
(Perkin-Elmer, Foster City, CA, USA). Computer-assisted analyses of DNA sequences
were carried out using the University of Wisconsin Genetics Computer Group (GCG)
package (Genetics Computer Group, INC., Madison, WI, USA), and the online services
available at the National Center of Biotechnology Information
(http://www.ncbi.nlm.nih.gov).
Nucleic acids
Total RNA was prepared by using the TRIzol reagent (Invitrogen) following the
manufacturer’s instructions. For Northern analysis, 30 µg of RNA were separated in
formaldehyde-agarose gels and transferred onto Nytran (Schleicher & Schuell)
membranes. 32 P-labelled probes were prepared using the Rediprime labelling kit
(Amersham) as recommended by the manufacturer. Hybridization and washing
conditions were performed as described in Church and Gilbert (1984).
RT-PCR and cloning of the PCR products
For reverse transcriptase reactions, we used 5 µg of total RNA obtained from
different tomato tissues, and the M-MLV reverse transcriptase (Promega). 5 µl of the
reverse transcriptase reaction were used for PCR, employing the following
oligonucleotide primers to specifically amplify JIP21: JIP21F (5’-